PROJECT SUMMARY/ABSTRACT Bacteria require fatty acids for a variety of biological functions, including the construction of phospholipids. Staphylococcus aureus, like other bacteria, synthesize fatty acids using a fatty acid biosynthesis pathway referred to as FASII, but can also scavenge fatty acids from the environment using the recently described Fak pathway. In addition to fatty acid biosynthesis and acquisition, it is common for bacteria to degrade fatty acids using fatty acid degradation (Fad) enzymes. In some cases, this is to adjust chain length but also occurs for ?-oxidation of fatty acids for energy generation. S. aureus does not perform ?-oxidation of fatty acids and has been thought to not possess the capacity to degrade fatty acids. This is due, in part, to the absence of a key crotonase enzyme. This is surprising considering that S. aureus is annotated to encode all the other necessary Fad enzymes, though their function has not been confirmed. During RNAseq studies of the Fak pathway, we observed an ~17-fold increased expression of the group of genes annotated to encode Fad proteins. Indeed, no crotonase enzyme was apparent, though all the other Fad functionalities were annotated. Using bioinformatics, we identified a putative crotonase enzyme in S. aureus that we now call FadB based on nomenclature in other systems. When expressed with S. aureus FabA, the S. aureus FadB can functionally complement an E. coli fadAB mutant on minimal media with fatty acid as a sole carbon source. This demonstrates that 1) S. aureus FadB can substitute for the E. coli crotonase activity-containing enzyme, and 2) S. aureus does possess a complete Fad pathway and likely can degrade fatty acids. We seek to characterize this pathway using two complementary Specific Aims. Aim 1 uses the E. coli model to confirm the identity of the other S. aureus Fad proteins and to define the activity of S. aureus FadB, including the substrate range in a well-described system with the benefit of minimal media. We found that under rich media conditions (there is no minimal media for S. aureus) that the S. aureus Fad pathway is lowly expressed and it is unknown if these genes are co-transcribed. Aim 2 determines the genetic context of the Fad-encoding genes. In addition, Aim 2 examines the ability for S. aureus to degrade fatty acids using controlled expression of the Fad genes by a combination of radio-labelled fatty acids and mass spectrometry approaches. The Fad-encoding genes have been identified in a variety of transcriptomic studies, but remain unstudied likely due to the known dogma in the field that S. aureus does not possess a complete Fad pathway and cannot degrade fatty acids. We anticipate that the completion of this application will redefine fatty acid metabolism in S. aureus and determine for the first time that S. aureus can degrade fatty acids. This will change how the field understands S. aureus metabolism and will set the stage for future applications examining the role of this pathway in cell physiology and pathogenesis.